Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, is responsible for two million deaths annually, and latently infects a third of the world's population. The threat of TB has increased in recent years because of the proliferation of multiple drug-resistant strains and the emergence of extensively drug-resistant strains. Mtb is an intracellular pathogen that has evolved mechanisms to overcome environmental changes (e.g. low O2, NO, and available nutrients) encountered during infection. A detailed biochemical characterization of these mechanisms of adaptation as well as a better understanding of their roles during infection are essential requirements for the development of new and effective anti-tubercular drugs. For instance, Mtb has the unusual ability to catabolize host cholesterol as a source of carbon and energy, and this capacity has been shown to be required for persistence in animal models. However, cholesterol catabolic activities, including the beta-oxidation reactions and the degradation of the aliphatic side-chain of cholesterol, are not fully understood. We and others showed that the degradation of the cholesterol side-chain led to changes in Mtb's lipidome, including an increased mass of the lipid virulence factor phthiocerol dimycocerosate. We also reported on the role of the two Mtb sterol C26-monooxygenases, CYP125 and CYP142, in the initiation of cholesterol side-chain degradation, and demonstrated that this activity was essential for growth of Mtb on cholesterol as a source of carbon and energy and for detoxification of cholest-4-en-3-one. Based on these observations, we hypothesize that the catabolism of the cholesterol side-chain during infection reshapes Mtb's metabolism as an adaptive mechanism to survive intracellularly. To test our hypothesis, in Specific Aim 1, we will identify essential and redundant enzymatic activities for the degradation of the cholesterol side-chain using Mtb knockout strains. In Specific Aim 2, metabolomic profiling and enzymatic assays will be conducted to identify the physiological substrate(s) of the cholesterol side-chain degrading enzymes. In Specific Aim 3, we will use an inducible protein knockdown approach to better define the role of cholesterol side-chain degrading enzymes in infectivity and persistence in vivo. The proposed study will extend the current knowledge of Mtb's mechanisms of adaptation for long-term survival and will help to validate new drug targets.